Ferritin fusion proteins for use in vaccines and other applications

ABSTRACT

An isolated ferritin fusion protein is provided in which ferritin is fused with a protein or peptide capable of being fused to ferritin without interfering with the polymeric self-assembly of the resulting fusion protein, and the protein may be of the endocapsid form when fused at the C terminus or an exocapsid form when fused at the N terminus. These fusion proteins may self-assemble into a variety of useful higher polymeric forms, e.g., capsid or other polymeric aggregate, and they are advantageous in that they are useful in a variety of applications, including human and veterinary vaccines and therapeutics, blood substitutes, image contrast agents, metal chelating agents, gelling agents, protein purification platforms, and therapeutic receptor-binding proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 10/435,666, filed May 12, 2003, which claimed thebenefit of U.S. provisional application 60/379,145, filed May 10, 2002,incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates in general to ferritin fusion proteins,and in particular to the fusion of additional protein or peptidesegments to either or both of the N and C termini, respectively, at theinner and outer surface of the ferritin protein to form a fusion proteincapable of self-assembly, and to the use of such fusion proteins asvaccines and in other applications including oxygen transport and thetherapeutic delivery of drugs and other therapeutic agents,

BACKGROUND OF THE INVENTION

Ferritin is a highly conserved 24 subunit protein that found in allanimals, bacteria, and plants. The major physiological function offerritin is to control the rate and location of polynuclear Fe(III)₂O₃formation (see, e.g., Theil, E. C. “The ferritin family of iron storageproteins,” Adv. Enzymol. Relat. Areas Mol. Biol. 63:421-449 (1990), andHarrison, P. M., Lilley, T. H. “Ferritin in Iron Carriers and IronProteins,” Loehr T. M., ed. Weinheim: VCH, 1990:353-452; these and allreferences cited in the present application are incorporated herein byreference). This control is achieved through biomineralization which isperformed by transporting hydrated iron ions and protons to and from amineralized core. Through this mechanism, ferritin accumulates iron atconcentrations orders of magnitude greater than the solubility of freeiron under physiological conditions. The rate of biomineralization isdirectly related to the ratio of ferritin H and L subunits (theso-called heavy and light chains) within each capsid and exhibits thegeneral trend of increasing the rate of iron storage with increasing Hchain content. These differences in capsid composition are tissuedependent and affect the mechanism of iron oxidation, core formation andiron turnover. For example, ferritin comprised of predominantly L chainis found in the serum, while ferritin from the heart has a high ferritinH content. The ferritin mineralized iron core acts to providebioavailable iron to a variety of redox enzymes and also serves adetoxification role.

Each ferritin protein is in the form of a 24 subunit capsid having 432symmetry, a diameter of 125 Å, a shell thickness of approximately 25 Åand a hollow inner core of approximately 80 Å in diameter (FIG. 1). Themonomeric ferritin typically has at least two isoforms denoted the L andH chains which differ in amino acid sequence. Although multiple forms ofH and L subunit lengths have been identified in many vertebratesincluding humans, these two forms are generally both found in theferritins that have been identified. Each ferritin subunit isapproximately a 17 kilodalton protein having the topology of a helixbundle which includes a four-antiparallel helix motif, with a fifthshorter helix (the C-terminal helix) lying roughly perpendicular to thelong axis of the 4 helix bundle. The helices are according to conventionlabeled ‘A, B, C, D & E’ from the N-terminus respectively. TheN-terminal sequence lies adjacent to the capsid three-fold axis andclearly extends to the surface, while the E helices pack together at thefour-fold axis with the C-terminus extending into the capsid core. Theconsequence of this packing creates two pores on the capsid surface. Thepore at the four-fold is approximately 4 to 5 Å across and predominantlyhydrophobic, while the three-fold pore, being slightly larger at 6.0 Ådiameter is predominantly hydrophilic. It is expected that one or bothof these pores represent the point by which the hydrated iron diffusesinto and out of the capsid.

Previous work on ferritins, such as disclosed in U.S. Pat. Nos.5,248,589; 5,358,722; and 5,304,382, all incorporated herein byreference, has focused on the physical aspects of the protein shell andthe core such that materials other than ferrihydrate may be locatedinside the shell. It has also been shown (S P Martsev, A P Vlasov, PArosio, Protein Engineering vol. 11, 377-381 (1998)) that recombinanthuman L and H ferritin when explored by differential scanningcalorimetry will dissociate into subunit monomers at pH 2.0 to 2.8.

Other recent works have involved the use of “virus-like” particles as amodular system for vaccines wherein antibody responses were induced inthe absence of adjuvants resulting in protection from viral infectionand allergic reactions (Lechner et al., Intervirology 2002; 45(4-6);212-7), but this system did not involved a ferritin-based development ofproteins. In Marchenko et al., J. Mol. Microbiol Biotechnol 2003;5(2):97-104, virus-like particles (VLPs) were constructed from a proteinknown as P1-380 which forms VLPs. In this case, fusion at the C and/orN-termini of the P1-380 protein did not interfere with the VLPself-assembly, and bi-functional fusion particles were made whichdemonstrated that they are more potent at generating and immuneresponse. Still further, Douglas et al. have performed some work whereina protein for the nucleation of iron was linked with the cowpea mosaicvirus (CCMV), See Adv. Mater., 14 (6):415-418 (2002). Still otherreferences refer to a “chimeric” protein using a virus-like particlewhich contains a nonstructural papillomavirus protein fused to the virusL2, a minor capsid protein. See Greenstone et al., PNAS USA, 95(4):1800-5 (1998). However, in all of these cases, these fusion proteins didnot involve ferritin.

Accordingly, none of the prior references have focused on utilizingferritin or the placement of the N and C-termini at the outer and innersurface of the capsid respectively (e.g., as shown in FIGS. 2A & B, anddescribed further below) for any purpose, and moreover, no one haspreviously has utilized this structure for the purpose of linkingsuitable proteins or peptides via fusion to ferritin in order to enhancethe properties of the proteins or peptides while creating a fusionprotein capable of self-assembly.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provideferritin fusion proteins which comprise proteins or peptide segmentscontiguously fused to ferritin, such at either or both of the N and Ctermini.

It is further an object of the present invention to provideferritin-fusion proteins further providing for a means to expressproteins which may be either incorporated onto the surface of thecapsid, or internalized through the extension of either terminus.

It is still further an object of the present invention to provideprotein fusion products which can be used in such applications asvaccines, therapeutics, image contrast agents, novel metal chelatingsystems, gelling agents, protein purification platforms, therapeuticreceptor-binding proteins, and other suitable applications.

It is even further an object of the present invention to provideferritin fusion proteins which can be used in human and veterinaryapplications as well as numerous non therapeutic applications.

It is another object of the present invention to provide ferritin fusionproteins with increased vascular residence times so as to improve thelikelihood of an immune response and provide prolonged therapeuticbenefits from drugs and other therapeutic agents.

It is yet another object of the present invention to provide recombinantferritin fusion proteins for use in vaccines, drug delivery, and manyother therapeutic methods involving proteins and peptide segments whichcan be fused to ferritin without interfering with the ability of theprotein for self-assembly or the ability to form higher polymericassemblies, such as a capsid structure or a polymeric aggregate.

These and other objects are provided by virtue of the present inventionwhich comprises a ferritin protein fused with a protein or peptide thatcan be expressed genetically along with the ferritin and which can allowthe formation of the polymeric assembly of the ferritin, such as aferritin capsid or other polymeric aggregate, in which the protein orpeptide is linked with the N or C terminal region of the ferritin. Theproteins or peptides will thus be used which do not restrict theself-assembly of the resulting fusion protein into useful higherpolymeric forms, e.g., the capsid form, but other polymeric forms suchas hemispherical shape, cylindrical, etc., are also possible. Inaccordance with the invention, the ferritin-fusion proteins provide ameans to express proteins which may be either incorporated onto theouter portion of the ferritin, e.g., on the surface of the capsid, orinternalized through the extension of either terminus. The advantages ofthe fusion proteins of the invention are manifold in that they caninclude viral envelope and capsid proteins so as to be utilized as viralvaccines, and because it is possible to have multiple proteins andpeptides incorporated into the fusion protein of the invention, it ispossible to construct multivalent fusion proteins, that can act asmultivalent vaccines, containing different proteins from the sameorganism, or proteins from different organisms.

In addition, when formed into the ferritin capsid structure in which theC-terminal region is located at the inner core of the ferritin proteinand the N-terminal region is located at the outer surface of theprotein, it will be possible to construct vaccines wherein one type ofprotein or peptide antigen is located on the surface of the ferritin andwill rapidly generate antibodies, but a second desired antigen can belinked at the internal C-terminal region and thus shield this antigenfrom initial immunogenic reaction for an extended period of time. Thevaccine will thus have an initial portion that generates an initial setof antibodies, and will have a second portion which becomes immunogeniconly after sufficient time has elapsed and the second antigen is exposedfollowing dissociation of the ferritin core. Such internal shielding canprovide a means to present non-aqueous soluble antigens. Even further,because the linkage with ferritin will enhance the useful lifetime ofthe protein or peptide before it is degraded, the fusion proteins of theinvention will be useful in extending the useful life and beneficialeffect of therapeutic proteins and peptides. Still other benefitspossible by virtue of the fusion proteins of the invention is the use ofthe human capsid (or animal capsid in veterinary applications) to avoidimmune-related problems when it is desired to make the linked peptide orprotein be less likely to generate an immune response. A further exampleis the use of the ferritin capsid to assemble human hemoglobin polymersfor use as potential oxygen transporting blood substitutes. Finally,such fusion proteins may also be beneficial in other ways, such as inmetal scavenging, encapsulating beneficial proteins or small molecules,or storing radioactive materials that may be combined with antibodiesand be targeted to a specific set of tissues or cells.

These embodiments and other alternatives and modifications within thespirit and scope of the disclosed invention will become readily apparentto those skilled in the art from reading the present specificationand/or the references cited herein, all of which are incorporated byreference.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is a ribbon diagram of ferritin capsid as viewed in the directionof the 4-fold axis (center). Subunits shown in alternating colors.

FIGS. 2A-2B show stereoviews illustrating the view of ⅓ of the ferritincapsid down a four-fold axis (center). The exterior N-terminus andinterior C-terminus are labeled clearly showing the availability of thetermini for the creation of recombinant fusion peptides or proteins.FIG. 2A shows the view from inside the capsid, and FIG. 2B the view fromthe exterior surface.

FIG. 3 is a schematic view of the plasmid coding for the fusion proteinof human alpha chain hemoglobin to human ferritin C-terminus inaccordance with the invention.

FIG. 4 is a stereo view of the packing around the 4-fold axis. Thearrows indicate the direction of the hypothetical rotation of subunitsto accommodate large C-terminal fusion products.

FIG. 5 illustrates the regularization histogram of (F_(L). G. Hα).

FIG. 6 illustrates the regularization histogram of native horse heartferritin.

FIG. 7 illustrates the regularization histogram of (F_(L). GG. Ag4).

FIG. 8 is a transmission electron microscopy picture showing the propercapsid formation of (F_(L). GG. Ag4).

FIG. 9 is a schematic view of the plasmid coding for the fusion proteinof HIV Tat protein (84 mer) to the human ferritin N-terminus inaccordance with the invention.

FIG. 10 shows the Western blot analysis using polyclonal antibodies toTat which positively identified the ferritin-Tat fusion protein of thepresent invention

FIG. 11 illustrates the regularization histogram of (Tat.6G.F_(L)).

FIG. 12 A is a schematic view of the plasmid coding for the fusionprotein of a small HIV Tat peptide to the human ferritin light chainN-terminus in accordance with the invention.

FIG. 12B is a schematic view of the plasmid coding for the fusionprotein of the HIV P24 protein to the human ferritin light chainN-terminus in accordance with the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, there are provided ferritinfusion proteins which comprise a fusion product between at least onechain of ferritin, such as the H or L chain, with a protein or peptidecapable of binding at the N terminus or C terminus of ferritin yet whichdoes not interfere with the ability of the resulting fusion protein toform a polymeric assembly, such as a capsid, a polymeric aggregate, orother functional shape. Ferritin is a highly conserved 24 subunitprotein that is found in all animals, bacteria, and plants which actsprimarily to control the rate and location of polynuclear Fe(III)₂O₃formation through the transportation of hydrated iron ions and protonsto and from a mineralized core. Through this mechanism, ferritinaccumulates iron at concentrations orders of magnitude greater than thesolubility of free iron under physiological conditions. The rate ofbiomineralization is directly related to the ratio of ferritin H and Lsubunits (the so-called heavy and light chains) within each capsid andexhibits the general trend of increasing the rate of iron storage withincreasing H chain content. These differences in capsid composition aretissue dependent and affect the mechanism of iron oxidation, coreformation and iron turnover. The ferritin mineralized iron core acts toprovide bioavailable iron to a variety of redox enzymes and also servesa detoxification role.

Each ferritin protein is in the form of a 24 subunit capsid having 432symmetry, a diameter of 125 Å, a shell thickness of approximately 25 Åand a hollow inner core of approximately 80 Å in diameter (FIG. 1). Themonomeric ferritin typically has at least two isoforms denoted the L andH chains which differ in amino acid sequence, and multiple forms of Hand L subunit lengths have been identified in many vertebrates includinghumans. Each ferritin subunit is approximately a 17 kilodalton proteinhaving the topology of a helix bundle which includes a four antiparallelhelix motif, with a fifth shorter helix (the c-terminal helix) lyingroughly perpendicular to the long axis of the 4 helix bundle. Thehelices are according to convention labeled ‘A, B, C, D & E’ from theN-terminus respectively. The N-terminal sequence lies adjacent to thecapsid three-fold axis and clearly extends to the surface, while the Ehelices pack together at the four-fold axis with the C-terminusextending into the capsid core. The consequence of this packing createstwo pores on the capsid surface. The pore at the four-fold isapproximately 4 to 5 Å across and predominantly hydrophobic, while thethree-fold pore, being slightly larger at 6.0 Å diameter ispredominantly hydrophilic. It is expected that one or both of thesepores represent the point by which the hydrated iron diffuses into andout of the capsid.

In accordance with the invention, suitable proteins or peptides can befused with the ferritin protein either as an exocapsid product by fusionwith the N-terminal sequence lying adjacent to the capsid three-foldaxis, as an endocapsid product by fusion with the C-terminus extendinginside the capsid core, or a combination thereof By ferritin is meantthe ferritin protein and/or its H and/or L chains as well as ferritinanalogs such as disclosed in U.S. Pat. No. 5,304,382, incorporatedherein by reference, and apoferritin, as well as those proteins havingthe structure of ferritin, namely an outer surface having a N-terminalregion and an inner core having an internal C-terminal region. Theproteins or peptides useful in the invention will include thoseproteins, peptides, antibodies, fragments, enzymes, haptens,peptidoglycans or other molecules including amino acid sequences whichcan be linked to ferritin, and which can link to ferritin withoutdisrupting its structure and which when expressed will form into aferritin fusion protein which will self assemble into a largemacromolecular or polymeric assembly, often pending the nature of thefusion products, with the same general physical structure andconfiguration (N terminal at the surface and C terminal in an innercore) as natural ferritin.

When designing the fusion product in accordance with the invention, itmay be necessary to consider including ‘spacer’ residues, such asglycine or other suitable amino acids, between each ferritin and theprotein or peptide fused to the ferritin. In general, a spacer willincrease the distance between the center of the ferritin and the linkedprotein or peptide which may be desirable, e.g., in cases wherein it isdesired to provide additional space between the ferritin portion of thefusion protein and the fused protein or peptide. This might arise incases wherein the ferritin is fused to an antigenic protein or peptideand it is desired to have the antigen more exposed so as to raiseantibodies such as in the case of vaccines. In addition, when the fusionprotein of the invention is formed by a linking of ferritin with anantibody, a spacer may be desirable to allow the antibody to seek andbind with a target with less steric hindrance from the ferritin portionof the fusion protein. In general, the larger the linked molecule, thegreater the need to have an adequate spacer. Accordingly, in the case ofthe fusion products of the invention, either endocapsid or exocapsidfusion product, one or more glycine (or other suitable amino acids)residues may be utilized if so desired to allow space for positioning oflarger proteins around the exterior of the capsid. Glycine is generallydesirable for this purpose since it can be used to create flexible‘tethers’ which can also easily adapt to an extended polypeptideconformation.

As one skilled in the art would recognize, depending on thephysiological or physical need, the desired protein or peptide may befused inside the ferritin when it is desired to shield the protein fromenvironmental factors which may, for example inactivate or otherwisecause degradation or cleavage, and may be fused outside the core when itis desired that the fused protein or peptide be unshielded such as whenmore rapid immunogenicity is desired. In addition, internal (C-terminal)or external (N-terminal) capsid fusion proteins may be used to formmixed capsids. For example, more than one antigenic protein or peptidecan be expressed on the surface as well as within the core. This couldbe used to insure both antibody response as well as cellular immunity.Additionally, multiple enzymes expressed in the same manner can be usedto create highly concentrated enzyme “factories” for multistepbiochemical pathways. Such chimeric multivalent ferritins can beachieved through multiple expression in the same vector or by capsiddissociation by known methods and reassociation of the desired productas a mixture.

Accordingly, the present invention makes use of the placement of the Nand C-termini at the outer and inner surface of the polymeric assemblyrespectively (FIGS. 2A & B), and allows for fusion proteins to beconstructed using proteins or peptides linked to one or both of thesesites. In the preferred embodiment, the ferritin fusion protein of theinvention is prepared in any suitable manner wherein at least oneprotein or peptide can be linked to ferritin without causing adisruption of the resulting polymeric assembly, that is the protein orpeptide and ferritin will stay linked while the fusion protein formsinto the final stable polymeric assembly, and the ferritin will retainits basic structure of an inner core and an outer surface, with thelinkage being either at the N terminal region at the outer ferritinsurface or the C terminal region in the inner core of the ferritin (orat both regions if so desired). In one desired embodiment in accordancewith the invention, the fusion protein will take on the polymeric capsidshape characteristic of ferritin. However, it is understood that thepropensity of the ferritin to self associate can be advantageous andtake on many different forms, and not just the capsid, and such formsmay be other types of a polymeric assembly such as a polymericaggregate, hemisphere, cylinder, etc. Self-assembly products which areformed in accordance with the invention by fusion with ferritin willstill have desired properties for many applications, such as vaccines,as set forth further below. This fusion protein of the invention may beconstructed using any suitable means that would be well known to one ofordinary skill in this art, such as recombinantly produced or producedunder conditions wherein the individual protein units will form into thefusion protein of the invention, e.g., via chemical or physical means offusion.

In accordance with the invention, the ferritin-fusion proteins will thushave expressed proteins which may be either incorporated onto the outerportion of the fusion protein, e.g., by linkage to the external Nterminus, or which will be internalized through linkage with the Cterminus. As set forth in more detail below, the functions of theprotein fusion products in accordance with the invention includeapplications as vaccines, therapeutics, image contrast agents, novelmetal chelating systems, gelling agents, protein purification platforms,therapeutic receptor-binding proteins, etc., and may be used in humanand veterinary applications as well as numerous non-therapeuticapplications.

As indicated above, the recombinant production of the ferritin fusionproteins of the present invention can take place using any suitableconventional recombinant technology currently known in the field. Forexample, molecular cloning a fusion protein, such as ferritin with asuitable protein such as the recombinant human hemoglobin alpha subunit,can be carried out via expression in E. coli with the suitable ferritinprotein, such as the human ferritin L-chain. In this process, thefull-length cDNA of Hemoglobin alpha was ligated to the C-terminal offerritin light chain gene via a glycine linker (FIG. 3) using PCR-basedmethods. Following this preparation of the gene, protein expression andisolation and/or purification can be achieved, form example, by firstverifying the coding sequence of the fusion protein (e.g.,ferritin/hemoglobin) so that it has the correct DNA sequence. Theconstruct may then be transformed into protein expression cellsBL21(DE3), grown to suitable size, e.g., OD 1.0 (600 nm) and induced at30 degree with 1 mM of IPTG to activate T7 promoter. In this process,cells are resuspended in B-PER buffer and sonicated for protein release.The resulting fusion protein may be isolated and/or purified, such asfrom the supernatant using appropriate chromatographic or other methods,such as Size Exclusive and Gel Filtration Ion Exchange chromatography.The protein may be confirmed using conventional Western blot tests usingsuitable polyclonal and monoclonal antibodies.

Once the fusion proteins of the invention have been constructed it ispossible to confirm capsid formation such as by the followingobservations:

1) the purified expression product eluted from size exclusion gelchromatography will have a retention factor consistent with a proteincomplex larger than native recombinant ferritin (ferritin MW, 408K);

2) light scattering experiments of the protein will show a monodispersedprotein with an estimated diameter of approximately twice that of nativeferritin (FIG. 6 and Table 2); and

3) Western blots using suitable polyclonal antibodies (e.g., in thespecific case above, from both human ferritin and hemoglobin alpha) willeach independently give positive results for the fusion product.

In accordance with the invention, the number of subunits in the fusionprotein of the invention may be considerably greater in this complexthan the 24 in native ferritin. This indicates that the capsid has aninherent ability to increase the angle of subunit-subunit packing andthat dimers may rotate to pack with the ‘B’ helices parallel across thetwo-fold axis, and are potentially further stabilized through theflexibility of the ‘Loop B-C’ surface loops which pack as anantiparallel beta sheet across the two-fold axis. This hypotheticalrotation could be encouraged by steric interactions, and thus aflattening of the capsid curvature would provide more accommodation ofthe large hemoglobin molecules. Small changes in these subunit packingangles could correlate with a great increase in capsid diameter andallow the incorporation of larger fusion products in the capsid core. Itis further understood that the modification or replacement of theexposed surface loop, Loop BC, could also be used to create ‘chimeric’ferritin molecules for vaccines and other applications.

The fusion proteins of the present invention may thus be utilized toenhance the properties of a number of proteins and peptides which areadministered internally for a therapeutic purpose. In particular,through linkage with ferritin, the therapeutic protein will have itshalf life in plasma greatly extended when fused with ferritin whichnormally has a half-life of 18-20 hours. Thus, a beneficial protein orpeptide will be able to continue providing therapeutic benefits longafter the non-fused protein or peptide would have been completeddegraded in the bloodstream. In addition, fusing the protein or peptideto ferritin may avoid immune related problems, especially in those caseswherein the fused protein is linked at the inner C-terminal region offerritin. Similarly, the fusion to ferritin may also protect certainproteins and peptides (e.g., enzymes, toxic chelated compounds or smallmolecule therapeutics) which would otherwise be rapidly dissolved in thebloodstream, and once again in these cases it is desirable to have thesepeptides and proteins linked to the C-terminal region of ferritin sothat they will fuse and be located in the inner encapsulated core of theferritin portion of the fusion protein.

Economical and Scalable Isolation and Purification of Ferritin FusionProducts

Still further, by fusing a protein or small peptide with an incorporatedenzyme cleavage site to the exterior of ferritin, the fusion product canbe easily isolated once cleaved due to the large size difference of theferritin capsid—simple ultra-filtration to isolate final product. Thusthe ferritin fusion platform can be used for the convenient andinexpensive isolation of exocapsid fusion products.

Precipitation of Metal Complexes

The propensity of the ferritin core to precipitate a variety of metalcomplexes, including certain ceramics (see, e.g., U.S. Pat. Nos.5,248,589; 5,358,722; and 5,304,382) in its natural state and given thatnovel metal nucleating peptides can be expressed in the core asillustrated in the enclosed Examples, it is understood that suchmetallic or inorganic complexes can be comprised of materials whichpromote the incorporation of radioactive elements, elements enhancingthe properties of x-ray or nuclear magnetic resonance contrast agents,that are beneficial for a variety of medically related therapeutic,diagnostic, or prophylactic applications. It is further understood thatby using the capsid architecture to advantage, precious or rare metalscan be concentrated and precipitated in the core (as in the case of Fenormally) and as such these specialized ferritins can be used to easilyisolate by means of fermentation processes with bacteria, yeast etc.expressing the protein desired or undesired inorganics. Recent interesthas been in the control of particle size for nanoparticle production ofsemiconductor materials.

Antibody Directed Therapeutic Virus-Like Particles (“VLPs”)

Exocapsid fusion products which are formed from a fragment (Fv) orgreater domain structure of an antibody can direct therapeutics ordiagnostics contained in the capsid or expressed on the surface, tospecialized locations. In such an embodiment, it will be possible tolink a protein or peptide containing an agent used to target or destroycell such as tumors with a ferritin linked with an antibody (or anactive region from an antibody such as an active fragment) which willallow the fusion protein to be directed to the target tissue (e.g., thetumor). Accordingly, in this manner capsids in accordance with theinvention which contain toxic proteins, radioactive elements, or otherdestructive agents can be targeted directly to cancerous tissue.

Hemoglobin-Based Blood Substitutes

Ferritin fusion products with hemoglobin can potentially be used asnovel blood substitutes. Potential advantages of such chimeric ferritinsinclude 1) increased vasculature residence time; 2) restrictedendothelial interaction limiting or eliminating the undesirable effectof binding nitrogen oxide; 3) encapsulated forms can protect hemoglobinsfrom undesirable oxidation of Fe; and 4) polymeric forms can preventdissociation of hemoglobin alpha chains from the beta chains.

Vaccines

The fusion proteins of the invention as described above, may also beutilized in the development of vaccines for active and passiveimmunization against infections, as described further below.

In a further embodiment, when linked to ferritin in a fusion protein inaccordance with the invention, antibodies may be used as a passivevaccine which will be useful in providing suitable antibodies to treator prevent infections.

As would be recognized by one skilled in this art, vaccines inaccordance with the present invention may be packaged for administrationin a number of suitable ways, such as by parenteral (i.e.,intramuscular, intradermal or subcutaneous) administration ornasopharyngeal (i.e., intranasal) administration. One such mode is wherethe vaccine is injected intramuscularly, e.g., into the deltoid muscle,however, the particular mode of administration will depend on the natureof the infection to be dealt with and the condition of the patient. Thevaccine is preferably combined with a pharmaceutically acceptablecarrier to facilitate administration, and the carrier is usually wateror a buffered saline, with or without a preservative. The vaccine may belyophilized for resuspension at the time of administration or insolution.

The preferred dose for administration of a fusion protein in accordancewith the present invention is that amount which will be effective inimmunizing a patient, i.e., in having that patient develop antibodiesagainst a general or specific condition. This amount is generallyreferred to as an “immunogenic amount”, and this amount will varygreatly depending on the nature of the antigen and of the immune systemand the condition of the patient. Thus an “immunogenic amount” of fusionprotein used in accordance with the active vaccines of the invention isintended to mean a nontoxic but sufficient amount of the antigenic agentsuch that the desired prophylactic or therapeutic generation ofantibodies is produced. Accordingly, the exact amount of the immunogenicagent that is required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe condition being treated, the particular carrier or adjuvant beingused and its mode of administration, and the like. Accordingly, the“immunogenic amount” of any particular fusion protein composition willvary based on the particular circumstances, and an appropriateimmunogenic amount may be determined in each case of application by oneof ordinary skill in the art using only routine experimentation. Thedose should be adjusted to suit the individual to whom the compositionis administered and will vary with age, weight and metabolism of theindividual. The compositions may additionally contain stabilizers orpharmaceutically acceptable preservatives, such as thimerosal(ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma ChemicalCompany, St. Louis, Mo.).

Accordingly, an active vaccine in accordance with the invention isprovided wherein an immunogenic amount of an isolated protein asdescribed above is administered to a human or animal patient in need ofsuch a vaccine. The vaccine may also comprise a suitable,pharmaceutically acceptable vehicle, excipient or carrier. In accordancewith the invention, it is thus possible to link ferritin with viralproteins, e.g., envelope proteins or other proteins from suchpotentially highly pathogenic viruses such as AIDS, SARS, etc., and thenuse the fusion proteins as a means of developing antibodies against theAIDS and/or SARS viruses. In addition to providing vaccines which may beprotective against such potentially deadly diseases, such fusionproteins may also be utilized in research concerning these diseases, andmay be useful in developing methods or drugs in addition to vaccineswhich can be effective against these diseases.

When the fusion proteins of the invention are linked with antibodies,these may be used in passive vaccines. In this case, the preferred dosefor administration of an antibody composition in accordance with thepresent invention is that amount will be effective in preventing oftreating an infection, and one would readily recognize that this amountwill vary greatly depending on the nature of the infection and thecondition of a patient. An “effective amount” of fused antibody to beused in accordance with the invention is intended to mean a nontoxic butsufficient amount of the antibody such that the desired prophylactic ortherapeutic effect is produced. Accordingly, the exact amount of theantibody or a particular agent that is required will vary from subjectto subject, depending on the species, age, and general condition of thesubject, the severity of the condition being treated, the particularcarrier or adjuvant being used and its mode of administration, and thelike. Accordingly, the “effective amount” of any particular antibodycomposition will vary based on the particular circumstances, and anappropriate effective amount may be determined in each case ofapplication by one of ordinary skill in the art using only routineexperimentation. The dose should be adjusted to suit the individual towhom the composition is administered and will vary with age, weight andmetabolism of the individual. The compositions may additionally containstabilizers or pharmaceutically acceptable preservatives, such asthimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (SigmaChemical Company, St. Louis, Mo.).

In addition, the antibody compositions of the present invention and thevaccines as described above may also be administered with a suitableadjuvant in an amount effective to enhance the immunogenic responseagainst the conjugate. For example, suitable adjuvants may include alum(aluminum phosphate or aluminum hydroxide), which is used widely inhumans, and other adjuvants such as saponin and its purified componentQuil A, Freund's complete adjuvant, and other adjuvants used in researchand veterinary applications. Still other chemically defined preparationssuch as muramyl dipeptide, monophosphoryl lipid A, phospholipidconjugates such as those described by Goodman-Snitkoff et al. J.Immunol. 147:410-415 (1991) and incorporated by reference herein,encapsulation of the conjugate within a proteoliposome as described byMiller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated byreference herein, and encapsulation of the protein in lipid vesiclessuch as Novasome™ lipid vesicles (Micro Vescular Systems, Inc., Nashua,N.H.) may also be useful.

Another, functional aspect of the ferritin when compared to other viruscapsid vaccines is that unlike a virus capsid which will be recognizedby the immune system quickly, when an endocapsid fusion product inaccordance with the present invention is used by itself, the capsid willnot be recognized as foreign until is begins to disassemble and theantigen becomes exposed. That means that one could create a time-releaseantigenic effect which could potentially produce a greater immunitysince exposure to the antigens will continue for a much longer period oftime. The ferritin fusion proteins are less complicated and potentiallymuch easier to make than virus-like ones, particularly those which havemore than one protein structural component of the capsid.

Pharmaceutical Compositions

As would be recognized by one skilled in the art, the fusion proteins ofthe present invention may also be formed into suitable pharmaceuticalcompositions for administration to a human or animal patient in order totreat or prevent infections, or to be used as therapeutic agents againstother diseases or conditions. Pharmaceutical compositions containing thefusion proteins of the present invention as defined and described abovemay be formulated in combination with any suitable pharmaceuticalvehicle, excipient or carrier that would commonly be used in this art,including such as saline, dextrose, water, glycerol, ethanol, othertherapeutic compounds, and combinations thereof. As one skilled in thisart would recognize, the particular vehicle, excipient or carrier usedwill vary depending on the patient and the patient's condition, and avariety of modes of administration would be suitable for thecompositions of the invention, as would be recognized by one of ordinaryskill in this art. Suitable methods of administration of anypharmaceutical composition disclosed in this application include, butare not limited to, topical, oral, anal, vaginal, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal and intradermaladministration.

For topical administration, the composition is formulated in the form ofan ointment, cream, gel, lotion, drops (such as eye drops and eardrops), or solution (such as mouthwash). Wound or surgical dressings,sutures and aerosols may be impregnated with the composition. Thecomposition may contain conventional additives, such as preservatives,solvents to promote penetration, and emollients. Topical formulationsmay also contain conventional carriers such as cream or ointment bases,ethanol, or oleyl alcohol.

Other Applications

As set forth above, in accordance with the invention, the ferritinfusion proteins can have a number of potential uses in both the area ofvaccines and other pharmaceutical and therapeutic compositions, as wellas in many other areas which can provide beneficial effects. Forexample, the ferritin fusion proteins of the invention may be used tostore radioactive metals in concentrated form which attached toantibodies can direct concentrated therapeutics to cancerous tissues. Inaddition, because of the potential ability of ferritin to bind iron andother precious metals, it may be possible to use the ferritin fusionproteins of the invention in systems wherein precious metals areobtained by scavenging methods, and this would provide an“Earth-friendly” mining operation since toxic chemicals could beavoided. In addition, since it appears that relative L and H chaincomposition may be involved in certain tissues, it is possible thatferritin fusion products having a specific proportion of L to H chains,or a predominant amount (e.g., 60-100%) of one type of chain may allowone to direct the capsids and therefore therapeutics (DNA, etc) tocertain tissues. For example, it appears that heart muscle tissuegenerally is characterized by ferritins having predominantly H chains,wherein ferritin in the bloodstream is generally found to havepredominantly L chains.

Still other applications include Macro structure assembly platform formore complicated systems—nano-technology applications. In addition,Ferritin, encapsulated therapeutics or other agents directed totherapeutic or other desired targets by attached antibodies or othermeans. In the case of antibodies, antibodies can be intact or possessonly the antigen recognition portions, such as the Fv fragment and canbe attached to ferritin by chemical or recombinant methods. It is alsopossible to modify through insertion various components of the ferritincapsid to produce hybrid molecules as vaccines and therapeutics. Forexample, the replacement of Loop BC located on the surface of theprotein. It is also contemplated that certain difficult-to-crystallizepeptides or proteins may be crystallized as the capsid—especially whenexpressed internally and thereby preserving the current exterior crystalpacking interactions. Internal expression may also improve thesolubility problems associated with certain hydrophobic proteins andpeptides. The ferritin fusion proteins may also be used in applicationswherein linkage will slow the rotation of a particle used in identifyingprocesses such as NMR, image contrast, or X-ray imaging, and thus thefusion proteins of the invention will be useful in these contexts aswell.

In short, the ferritin fusion proteins of the present invention asdescribed above can be extremely useful in vaccines and otherpharmaceutical and therapeutic compositions, and will have particularuse in other applications such as drug delivery, oxygen transport, andother applications wherein enhancement of vascular residence time isdesired.

EXAMPLES

The following examples are provided which exemplify aspects of thepreferred embodiments of the present invention. It should be appreciatedby those of skill in the art that the techniques disclosed in theexamples which follow represent techniques discovered by the inventorsto function well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Example 1 Endocapsid Fusion

Recombinant Fusion of Human Alpha chain Hemoglobin to the Human FerritinC-Terminus Via a Single Glycine Spacer Sequence.

Capsid Abbreviation: (F_(L). G. Hα).

Molecular cloning: Recombinant human hemoglobin alpha subunit wasexpressed in E. coli as a human ferritin L-chain fusion protein. Thefull-length cDNA of Hemoglobin alpha was ligated to the C-terminus offerritin light chain gene via a glycine linker (FIG. 3) using the PCRbased method.

Protein expression and purification: Coding sequence ofFerritin/hemoglobin was verified by DNA sequence. The construct wastransformed into protein expression cells BL21(DE3). The transformedcells were grown to OD 1.0 (600 nm) and induced at 30 degree with 1 mMof IPTG to activate the T7 promoter. Cells were resuspended in B-PERbuffer and sonicated for protein release. Recombinant fusion protein waspurified from supernatant using Size Exclusive and Gel Filtration IonExchange chromatography. The protein was confirmed with Western blotusing both polyclonal and monoclonal antibodies.

Capsid or self assembled particle (SAP) formation was indicated by thefollowing observations:

1) the purified expression product eluted from size exclusion gelchromatography with a retention factor consistent with a protein complexlarger than native recombinant ferritin (ferritin MW, 408K);

2) light scattering experiments of the protein shown in FIG. 5 & Table1, indicated a monodispersed protein with an estimated diameter ofapproximately 2.5 times the size of native ferritin based on the valuesshown in FIG. 6 & Table 2 (these values are generally not accurate, butevidence for monodispersity are important in providing strong evidencefor a uniform size and potentially ordered SAP); and 3) Western blotsusing polyclonal antibodies from both human ferritin and hemoglobinalpha each independently gave positive results for the fusion product.

The number of subunits implied by the light scattering results isconsiderably greater in this complex than the 24 in native ferritin.While the exact configuration of the complex is currently unknown, theSAP is homogenous in nature consistent with a single molecular entity.These observations suggest that the subunit-subunit association has aninherent ability to increase the angle of packing. It is postulated thatthe dimers (shown in FIG. 4) rotate to pack with the ‘B’ helicesparallel across the capsid two-fold axes, an interaction potentiallyfurther stabilized through the flexibility of the ‘Loop B-C’ surfaceloops which pack as an antiparallel beta sheet across the two-fold axes.This hypothetical rotation could be encouraged by the stericinteractions between the hemoglobin alpha chain, i.e., a flattening ofthe capsid curvature would provide more accommodation of the largehemoglobin molecules. Small changes in these subunit packing anglescould correlate with a great increase in capsid diameter and allow theincorporation of larger fusion products in the capsid core. TABLE 1Cumulants datalog of F_(L).G.Hα. Data collected on a ProteinsolutionsDynapro light scattering spectrophotometer at 22 C. Meas. # Time (s) AmpDiff Rad (nm) MW Polyd. (nm) Temp (C.) Count Rate Base Line SOS 1 10.000.5464 60.32 34.82 1.36E+04 23.52 20.0 1509998 1.0000 34.77 2 20.130.5379 61.50 34.15 1.30E+04 19.03 20.0 1557511 1.0000 24.08 3 30.260.5227 59.83 35.10 1.39E+04 21.33 20.0 1487880 0.9998 30.99 4 40.390.5142 61.69 34.04 1.29E+04 23.51 20.0 1558383 0.9974 27.37 5 50.530.5378 56.64 37.08 1.58E+04 26.54 20.0 1595309 0.9983 35.11 6 60.660.5393 60.72 34.59 1.34E+04 18.28 20.0 1498222 0.9998 18.44 7 70.790.5370 62.29 33.72 1.27E+04 24.31 20.0 1472016 1.0020 29.79 8 80.930.5499 58.83 35.70 1.45E+04 24.14 20.0 1566428 1.0030 30.84 9 91.060.5260 60.41 34.77 1.36E+04 19.45 20.0 1560468 0.9970 23.29 10 101.200.5329 60.25 34.86 1.37E+04 15.90 20.0 1573030 1.0020 32.37 11 111.300.5420 56.99 36.85 1.56E+04 26.87 20.0 1588833 0.9981 43.30 12 121.500.5432 58.73 35.76 1.45E+04 18.02 20.0 1582105 1.0010 29.54 13 131.600.5534 57.88 36.28 1.50E+04 23.24 20.0 1512688 0.9984 31.32 14 141.700.5516 60.52 34.70 1.35E+04 18.63 20.0 1492581 1.0060 34.87 15 151.900.5522 60.71 34.59 1.34E+04 18.58 20.0 1466885 1.0040 28.24 16 162.000.5625 59.91 35.05 1.39E+04 19.16 20.0 1513441 0.9983 28.34 17 172.100.5563 60.32 34.81 1.36E+04 16.63 20.0 1544321 0.9965 34.78 18 182.300.5470 56.92 36.89 1.56E+04 26.16 20.0 1595394 1.0000 39.68 19 192.400.5494 55.77 37.66 1.64E+04 24.23 20.0 1589674 1.0010 29.99 20 202.500.5540 60.85 34.52 1.34E+04 19.87 20.0 1538082 1.0020 19.80 21 212.700.5635 60.34 34.81 1.36E+04 18.32 20.0 1509841 1.0010 35.23 22 222.800.5771 58.34 36.00 1.47E+04 19.37 20.0 1535249 1.0020 32.82

TABLE 2 Cumulants datalog of native horse heart ferritin. Data collectedon a Proteinsolutions Dynapro light scattering spectrophotometer at 22C. Meas. # Time (s) Amp Diff Rad (nm) MW Polyd. (nm) Temp (C.) CountRate Base Line SOS 1 10.00 0.3740 163.3 12.86 1327 6.238 20.0 44621410.9999 13.34 2 20.14 0.3624 158.7 13.23 1417 8.482 20.0 4476535 0.998515.93 3 30.27 0.3517 159.2 13.20 1409 9.485 20.0 4438350 0.9987 14.73 440.40 0.3458 158.9 13.22 1415 5.018 20.0 4446134 1.0000 14.73 5 50.540.3486 158.7 13.23 1418 5.580 20.0 4412028 1.0010 12.01 6 60.67 0.3434160.2 13.11 1387 7.886 20.0 4401034 0.9996 12.75 7 70.81 0.3440 157.613.33 1442 7.896 20.0 4413116 1.0000 12.36 8 80.94 0.3422 159.0 13.211412 4.990 20.0 4372660 0.9990 12.51 9 91.08 0.3455 155.8 13.48 14818.400 20.0 4376544 1.0010 13.24 10 101.20 0.3402 155.2 13.53 1494 7.22620.0 4447649 1.0010 12.63 11 111.30 0.3392 155.3 13.53 1492 8.702 20.04496696 0.9995 13.41 12 121.50 0.3421 153.5 13.68 1533 8.607 20.04460202 0.9991 14.85 13 131.60 0.3426 153.7 13.66 1528 9.477 20.04415901 0.9997 14.45 14 141.70 0.3368 156.2 13.44 1471 8.809 20.04380702 1.0000 18.02 15 151.90 0.3418 157.1 13.37 1453 6.909 20.04409014 0.9994 10.54 16 162.00 0.3406 155.0 13.55 1498 8.586 20.04369267 0.9998 11.70 17 172.10 0.3406 157.0 13.37 1453 7.102 20.04462261 0.9999 11.06 18 182.30 0.3416 155.3 13.53 1492 8.242 20.04390592 1.0010 12.73 19 192.40 0.3387 153.7 13.66 1528 8.784 20.04465543 1.0010 14.06 20 202.50 0.3386 156.3 13.44 1470 6.635 20.04451312 0.9998 10.17

Example 2 Endocapsid Fusion

Recombinant Fusion of Silver Condensing Peptide to the C-Terminus ofHuman L chain Ferritin Via a Two Glycine Spacer Sequence.

Capsid Abbreviation: (F_(L).GG.Ag4), AG4 is NPSSLFRYLPSD (Seq. ID No.1)

The proper capsid formation, as an example of a metal scavenging peptidein combination with ferritin, was indicated by the followingobservations:

1) the purified expression product eluted from size exclusion gelchromatography with a retention factor consistent with the nativerecombinant ferritin (MW, 408K); 2) light scattering experiments of theprotein shown in FIGS. 7 & Table 3 indicating a mono-dispersed proteinwith an estimated diameter of approximately 180 Å; 3) the silvercondensing properties of the capsid were confirmed; and 4) TEM imagesindicated a polyhedral capsid with the proper external dimensions moreconsistent with the x-ray structure of ferritin (FIG. 8). TABLE 3Cumulants datalog of (F_(L).GG.Ag4). Data collected on aProteinsolutions Dynapro light scattering spectrophotometer at 22 C.Meas. # Time (s) Amp Diff Rad (nm) MW Polyd. (nm) Temp (C.) Count RateBase Line SOS 1 10.00 0.3452 226.1 9.289 619.4 2.372 20.0 4053655 0.99982.853 2 20.14 0.3582 219.0 9.588 667.1 3.015 20.0 4093250 0.9997 2.641 330.27 0.3638 221.2 9.496 652.2 3.804 20.0 4065981 0.9992 3.905 4 40.400.3767 221.3 9.492 651.6 4.188 20.0 4055590 1.0000 4.852 5 50.54 0.4124220.2 9.537 658.9 4.272 20.0 4039832 0.9996 7.010 6 60.67 0.4178 218.59.610 670.7 3.011 20.0 4095640 1.0010 5.197 7 70.80 0.4196 221.5 9.482650.0 3.497 20.0 3961934 0.9998 5.097 8 80.94 0.4201 217.6 9.652 677.53.902 20.0 4038216 1.0000 4.480 9 91.07 0.4234 216.9 9.682 682.6 2.97720.0 3996752 1.0000 5.728 10 101.20 0.4239 221.2 9.493 651.7 3.519 20.03967679 1.0000 3.414 11 111.30 0.4369 220.2 9.537 658.9 3.485 20.03942308 1.0000 3.742 12 121.50 0.4388 216.4 9.705 686.3 2.274 20.03923822 1.0000 4.641 13 131.60 0.4371 219.7 9.559 662.5 3.618 20.03928869 1.0010 4.548 14 141.70 0.4436 216.2 9.714 687.9 2.800 20.03998526 0.9994 5.508 15 151.90 0.4400 217.7 9.648 677.0 3.565 20.03961948 0.9995 3.895 16 162.00 0.4403 215.9 9.729 690.3 2.484 20.03973764 1.0000 5.826 17 172.10 0.4363 223.1 9.413 639.0 2.679 20.03928356 1.0000 4.784 18 182.30 0.4332 220.6 9.520 656.1 3.814 20.03978646 1.0000 4.470 19 192.40 0.4282 220.8 9.511 654.6 1.427 20.03999961 1.0000 2.819 20 202.50 0.4259 220.0 9.547 660.5 4.180 20.03994938 1.0010 4.707

Example 3 Exocapsid Fusion

Recombinant Fusion of HIV Tat Protein (84 mer) to the N-Terminus Via aSix (6) Glycine Spacer Sequence.

Capsid Abbreviation: (Tat .6G.F_(L))

Where:

HIV Tat Sequence is (SEQ ID NO: 2) MEPVDPRLEP WKHPGSQPKT ACTNCYCKKCCFHCQVCFIT KALGISYGRK KRRQRRRAHQ NSQTHQASLS KQPTSQPRGD PTGPKE -

Glycine Spacer is GGGGGG (SEQ ID NO: 3)

Human ferritin L chain sequence is (SEQ ID NO: 4) MSSQIRQNYS TDVEAAVNSLVNLYLQASYT YLSLGFYFDR DDVALEGVSH FFRELAEEKR EGYERLLKMQ NQRGGRALFQDIKKPAEDEW GKTPDAMKAA MALEKKLNQA LLDLHALGSA RTDPHLCDFL ETHFLDEEVKLIKKMGDHLT NLHRLGGPEA GLGEYLFERL TLKHD

Molecular cloning: Recombinant wild type HIV-1 Tat was expressed in E.coli as a human ferritin L-chain fusion protein. The full-length cDNA ofTat was ligated to the N-terminus of the ferritin light chain gene withsix Glycine linkers (FIG. 9) using the PCR based method.

Protein expression and purification: Coding sequence of Ferritin/Tat wasverified by DNA sequence. The construct was transformed into proteinexpression cells BL21(DE3). The transformed cells were grown to OD 1.0(600 nm) and induced at 30 degree with 1 mM of IPTG to activate T7promoter. Cells were resuspended in B-PER buffer and sonicated forprotein release. Recombinant fusion protein was purified fromsupernatant using Size Exclusive and Gel Filtration Ion Exchangechromatography. The protein was confirmed with Western blot usingpolyclonal and monoclonal antibodies (FIG. 10).

The proper capsid formation was indicated by the followingobservations: 1) the purified expression product eluted from sizeexclusion gel chromatography with a retention factor consistent with aprotein on the order or larger than native recombinant ferritin (MW,408K); 2) light scattering experiments of the protein shown in FIG. 11 &Table 4 indicating a mono-dispersed protein with an estimated diameterroughly twice that of native ferritin; and 3) Western blots usingpolyclonal antibodies to Tat gave positive results for the fusionproduct (FIG. 10). TABLE 4 Cumulants datalog of (Tat.6G.F_(L)). Datacollected on a Proteinsolutions Dynapro light scatteringspectrophotometer at 22 C. Meas. # Time (s) Amp Diff Rad (nm) MW Polyd.(nm) Temp (C.) Count Rate Base Line SOS 1 10.00 0.5372 64.68 32.471.16E+04 10.44 20.0 1056642 1.0030 10.32 2 20.14 0.5653 62.18 33.771.27E+04 11.77 20.0 1125065 0.9980 13.08 3 30.27 0.5784 61.52 34.141.30E+04 18.86 20.0 1111305 1.0000 20.11 4 40.40 0.5783 63.19 33.231.22E+04 15.06 20.0 1089854 0.9990 12.48 5 50.53 0.5717 64.18 32.721.18E+04 14.12 20.0 1095614 0.9972 10.08 6 60.67 0.5722 65.13 32.241.14E+04 7.92 20.0 1089823 0.9985 15.30 7 70.80 0.5740 64.68 32.471.16E+04 11.84 20.0 1089562 1.0010 9.33 8 80.93 0.5776 64.67 32.481.16E+04 14.45 20.0 1064212 1.0010 11.53 9 91.07 0.5833 63.48 33.081.21E+04 16.00 20.0 1087651 0.9993 16.48 10 101.20 0.5882 63.81 32.911.20E+04 15.88 20.0 1078616 0.9999 17.03 11 111.30 0.5846 63.55 33.051.21E+04 14.90 20.0 1059146 0.9973 15.79 12 121.50 0.6005 63.42 33.121.21E+04 14.67 20.0 1071728 0.9973 14.46 13 131.60 0.5982 62.87 33.401.24E+04 11.11 20.0 1112817 0.9993 9.72 14 141.70 0.6022 63.26 33.201.22E+04 10.18 20.0 1104251 1.0000 10.96 15 151.90 0.5945 65.18 32.221.14E+04 7.67 20.0 1113225 1.0020 5.47 16 162.00 0.5957 64.45 32.581.17E+04 11.57 20.0 1111261 1.0020 8.75 17 172.10 0.5880 64.25 32.691.18E+04 10.34 20.0 1124944 1.0000 15.35 18 182.30 0.6006 65.29 32.171.13E+04 7.27 20.0 1131531 0.9996 10.66 19 192.40 0.6046 64.26 32.681.18E+04 12.58 20.0 1097216 1.0000 14.31 20 202.60 0.6030 63.37 33.141.22E+04 13.85 20.0 1118774 1.0010 12.03

Example 4 Exocapsid Fusion

Recombinant Fusion of a Small HIV Tat Peptide to Human L Chain Ferritinwith a Six (6) Glycine Spacer Sequence

Capsid Abbreviation: (TatP.6G.FL) where TatP is QPKTACTNC (SEQ ID NO:5)

Molecular cloning: Recombinant wild type HIV-1 Tat peptide was expressedin E. coli as a human ferritin L-chain fusion protein. The full-lengthcDNA of Tat was ligated to the N-terminus of the ferritin light chaingene with six Glycine linkers (FIG. 12A) using a PCR based method.

Protein expression and purification: Coding sequence of Ferritin/Tatpeptide was verified by DNA sequence. The construct was transformed intoprotein expression cells BL21(DE3). The transformed cells were grown toOD 1.0 (600 nm) and induced at 30 degree with 1 mM of IPTG to activateT7 promoter. Cells were resuspended in B-PER buffer and sonicated forprotein release. Recombinant fusion protein was purified fromsupernatant using Size Exclusive and Gel Filtration Ion Exchangechromatography. In this case the protein did not produce a positiveWestern blot using polyclonal and monoclonal antibodies, presumably dueto the small size of the fusion peptide.

The proper capsid formation was indicated by the following observations:

1) the purified expression product eluted from size exclusion gelchromatography with a retention factor consistent with the nativerecombinant ferritin (MW, 408K).

Example 5 Exocapsid Fusion

Recombinant Fusion of HIV P24 Protein to the N-Terminus Via a Six (6)Glycine Spacer Sequence. (P24.6G. F_(L))

Molecular cloning: Recombinant wild type HIV-1 P24 was expressed in E.coli as a human ferritin L-chain fusion protein. The full-length cDNA ofTat was ligated to N-terminus of the ferritin light chain gene with sixGlycine linkers (FIG. 12B) using the PCR based method.

Protein expression and purification: Coding sequence of Ferritin/P24 wasverified by DNA sequence. The construct was transformed into proteinexpression cells BL21(DE3). The transformed cells were grown to OD 1.0(600 nm) and induced at 30 degree with 1 mM of IPTG to activate T7promoter. Cells were resuspended in B-PER buffer and sonicated forprotein release. Recombinant fusion protein was purified fromsupernatant using Size Exclusive and Gel Filtration Ion Exchangechromatography. The protein may have a truncated P24 component (pendingverification of expressed product). However, the resulting capsid fusionprotein reacts to give a positive Western blot using polyclonalantibodies.

The proper capsid formation was indicated by the following observations:

1) the purified expression product eluted from size exclusion gelchromatography with a retention factor consistent with the nativerecombinant ferritin MW, 408K; 2) Protein fusion product reacts to givea positive Western Blot using polyclonal P24 antibodies.

1. A ferritin fusion protein comprising a fusion protein selected fromthe group consisting of a ferritin protein fused at the C terminus witha protein or peptide capable of being fused to ferritin withoutinterfering with the polymeric assembly of the resulting fusion proteinand a ferritin protein fused at the N terminus with a protein or peptidecapable of being fused to ferritin without interfering with thepolymeric assembly of the resulting fusion protein.
 2. The ferritinfusion protein according to claim 1 wherein the fusion protein forms apolymer aggregate.
 3. The ferritin fusion protein according to claim 1wherein the fusion protein forms a capsid assembly.
 4. The ferritinfusion protein according to claim 1 wherein the protein is connected tothe ferritin by means of a spacer comprising at least one amino acid. 5.The ferritin fusion protein according to claim 4 wherein the amino acidis glycine.
 6. The ferritin fusion protein according to claim 5 whereinthe glycine spacer has from one to six glycine units.
 7. The ferritinfusion protein according to claim 1 wherein the protein fused toferritin is selected from the group consisting of hemoglobin, silvercondensing peptide, the HIV Tat protein, the small HIV Tat peptide,HIV-1 P24 protein, and viral proteins from the SARS or AIDS viruses. 8.The ferritin fusion protein according to claim 1 wherein the proteinfused to ferritin is selected from the group consisting of SEQ ID NO: 1,SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:
 7. 9. The ferritinfusion protein according to claim 1 wherein the ferritin is selectedfrom the group consisting of the ferritin L chain and the ferritin Hchain.
 10. The ferritin fusion protein according to claim 1 wherein theferritin is predominantly comprised of the ferritin L chain.
 11. Theferritin fusion protein according to claim 1 wherein the ferritin ispredominantly comprised of the ferritin H chain.
 12. The ferritin fusionprotein according to claim 1 that is prepared using recombinant means.13. An immunogenic composition comprising an immunogenic amount of thefusion protein according to claim
 1. 14. A pharmaceutical compositioncomprising the fusion protein according to claim 1 and apharmaceutically acceptable vehicle, carrier or excipient.
 15. Thecomposition according to claim 14 which is suitable for parenteral,oral, intranasal, subcutaneous, aerosolized or intravenousadministration in a human or animal.
 16. A method of eliciting animmunogenic reaction in a human or animal comprising administering tosaid human or animal an immunologically effective amount of an isolatedfusion protein according to claim
 1. 17. An isolated nucleic acidsequence coding for the fusion protein according to claim 1, ordegenerates thereof.
 18. A method of enhancing the half-life of proteinor peptide when internally administered to humans or animals comprisingforming said protein or peptide into a fusion protein with ferritinwherein the fusion protein maintains the same ability to self assembleas ferritin.
 19. A method of metal scavenging comprising introducing thefusion protein of claim 1 having a metal scavenging peptide fused toferritin into a fluid containing the metals to be scavenged for a timesufficient to allow the metal to bond with the metal scavenging peptideof the fusion protein, and then recovering the fusion protein having themetal bound thereto.
 20. A ferritin fusion protein comprising a fusionprotein selected from the group consisting of a ferritin protein fusedat the C terminus with a protein or peptide capable of being fused toferritin without interfering with the polymeric assembly of theresulting fusion protein and a ferritin protein fused at the N terminuswith a protein or peptide capable of being fused to ferritin withoutinterfering with the polymeric assembly of the resulting fusion protein,wherein the protein or peptide fused to ferritin is an immunogenicprotein or peptide capable of being fused to ferritin withoutinterfering with the polymeric assembly of the resulting fusion protein.21. A pharmaceutical composition comprising the fusion protein accordingto claim 20 and a pharmaceutically acceptable vehicle, carrier orexcipient.
 22. The composition according to claim 21 which is suitablefor parenteral, oral, intranasal, subcutaneous, aerosolized orintravenous administration in a human or animal.
 23. A vaccinecomprising an immunogenic amount of the fusion protein according toclaim 22 and a pharmaceutically acceptable vehicle, carrier orexcipient.
 24. A method of eliciting an immunogenic reaction in a humanor animal comprising administering to said human or animal animmunologically effective amount of the vaccine according to claim 23.25. A vaccine comprising an immunogenic amount of the fusion proteinaccording to claim 1 and a pharmaceutically acceptable vehicle, carrieror excipient.